EA031874B1 - Method of treating fabry disease - Google Patents

Abstract

The invention provides a method of treating Fabry disease in a subject, comprising administering to about 50 to about 600 mg of 1-deoxygalactone-dogyrimycin and an effective amount of a therapeutic agent to replace the α-Gal A deficiency of the enzyme, in which 1-deoxy-galactonogirimycin is administered from about 2 up to about 30 minutes before administering a therapeutic agent to replace the deficiency of the α-Gal A enzyme, while administering a therapeutic agent to replace the deficiency of the α-Gal A enzyme follows the introduction 1-deoxygalactone digestive.

Description

The present application provides a method for treating Fabry disease using 1-deoxyhalactonojirimycin and enzyme replacement therapy.

Background

Fabry disease is a progressive X chromosome-linked congenital metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) as a result of mutations in the α-Gal A gene (GLA). Despite the fact that the disorder is associated with Chromosome, women can express various degrees of clinical manifestations. Fabry disease is a rare disease with a number of cases estimated from 1 in 40,000 men to 1 in 117,000 of the general population. In addition, there are variants of the phenotype of Fabry disease with a late onset, which can be poorly diagnosed, since they do not manifest themselves with classical signs and symptoms. This, as well as a study of newborn screening for Fabry disease, suggests that the actual incidence of Fabry disease may be higher than currently estimated.

The clinical presentation of the disease can correlate with residual levels of α-Gal A. Without treatment, the life expectancy of patients with Fabry disease decreases, and death usually occurs at 40-50 years due to vascular diseases affecting the kidneys, heart and / or central nervous system. Enzyme deficiency leads to intracellular accumulation of the globotriaosylceramide (GL-3) substrate in the vascular endothelium and visceral tissues throughout the body. The gradual deterioration of renal function and the development of azotemia due to the accumulation of glycospingolipid usually occurs in 30-50 years, but can occur as early as 20 years. Renal lesions are found in both hemizygous (men) and heterozygous (women) patients.

Heart disease occurs in most men and many women. Early cardiac abnormalities include left ventricular dilatation, valve damage, and conduction disturbance. Mitral valve insufficiency is the most common valve disease, usually manifested in childhood or adolescence. Cerebrovascular manifestations are primarily the result of multifocal lesions of small vessels and may include thrombosis, transient cerebrovascular accidents, ischemia and basilar artery aneurysm, convulsions, hemiplegia, hemianesthesia, aphasia, labyrinth disorders or intracerebral hemorrhages. The average age at onset of cerebrovascular manifestations is 33.8 years. Personality changes and psychotic behavior can manifest as age increases.

A modern approved treatment for Fabry disease is enzyme replacement therapy (ERT). Currently, two α-Gal A products are available for treating Fabry disease: agalsidase alpha (Replagal®, Shire Human Genetic Therapies) and agalsidase beta (Fabrazyme®; Genzyme Corporation). These two forms of ERT are designed to compensate the patient for a lack of α-Gal A activity using a recombinant form of the enzyme administered intravenously. Although ERT is effective in many situations, treatment also has limitations. ERT has not shown a reduced risk of stroke, the heart muscle responds slowly, and the elimination of GL-3 from some types of kidney cells is limited. Some patients develop an immune response to ERT.

1-deoxygalactone jogrimycin and its salt, 1-deoxygalactone jogrimycin hydrochloride (also known as the U.S. Nomenclature of Medicines as Migalastat hydrochloride) act as a pharmacological chaperone for mutant α-Gal A by selective binding to the enzyme, thereby increasing its stability and contributing to the laying of the enzyme in its correct three-dimensional configuration. Such stabilization of α-Gal A allows cellular mechanisms of quality control to recognize the enzyme as correctly laid, as a result of which the transfer of the enzyme to the lysosome is increased, allowing it to perform its assigned biological function, GL-3 metabolism. As a result of the restoration of the correct transfer of α-Gal A from ER to the ligosome of migalastat hydrochloride also reduces the accumulation of improperly folded protein in ER, which can ease the load on the cells and some reactions like inflammation, which may be factors contributing to Fabry disease. Many in vitro and in vivo preclinical studies have been conducted, as well as clinical studies of migalastat hydrochloride. Migalastat hydrochloride has been shown to increase the amount of intracellular protein α-Gal A and enhances the transport of the mutant enzyme to the lysosome.

Short description

The present application provides a method for treating Fabry disease in a subject, comprising administering from about 50 to about 600 mg of 1-deoxyhalactonojirimycin and an effective amount of a therapeutic agent to replace a deficiency of α-Gal A enzyme in which 1-deoxyhalactonojirimycin is administered from about 2 hours to about 30 minutes before the introduction of a therapeutic agent to replace the deficiency of α-Gal A enzyme, while the introduction of a therapeutic agent to replace the deficiency of α-Gal A enzyme is responsible for the administration of 1 deoxygalactone-jirimycin.

In a specific embodiment, the patient abstains from eating for a period of time that begins about 0.5 to about 4 hours before the administration of 1-deoxyhalactone-jirimycin and ends about 0.5 to about 4 hours after the administration of 1-deoxyhalactone-jirimycin.

In another specific embodiment, the patient abstains from eating for at least about 2 hours before the administration of 1-deoxyhalactone-jirimycin and at least about 2 hours after the administration of 1-deoxyhalactone-jirimycin.

In another specific embodiment, 1-deoxyhalactonojirimycin is administered about 2 hours before the administration of a therapeutic agent to replace the deficiency of the α-Gal A enzyme.

In another specific embodiment, 1-deoxyhalactonojirimycin is migalastat hydrochloride.

In another specific embodiment, the therapeutic agent to replace the deficiency of the α-Gal A enzyme is selected from agal sidase alpha and agal sidase beta.

In another specific embodiment, 1-deoxyhalactonojirimycin is administered as an adjuvant to a therapeutic agent to replace the deficiency of the α-Gal A enzyme.

In another specific embodiment, 1-deoxyhalactonojirimycin and a therapeutic agent to replace an α-Gal A enzyme deficiency are administered as a combination therapeutic agent.

In another specific embodiment, the amount of 1-deoxyhalactonojirimycin administered is from about 150 mg to about 450 mg.

In another specific embodiment, the amount of 1-deoxyhalactonojirimycin administered is selected from about 150 mg, about 300 mg, and about 450 mg.

In another specific embodiment, a second dose of 1-deoxyhalactonojirimycin is additionally administered in the period from the moment the therapeutic agent is administered to replace the deficiency of the α-Gal A enzyme until about 4 hours after that.

In another specific embodiment, 1-deoxyhalactonojirimycin and a therapeutic agent to replace the deficiency of α-Gal A enzyme are administered every 1 to 4 weeks.

In another specific embodiment, 1-deoxyhalactonojirimycin and a therapeutic agent to replace the deficiency of α-Gal A enzyme are administered every 2 weeks.

A brief description of the graphic materials

In FIG. 1 shows the combination of plasma α-Gal A activities in patients who were treated for periods 1 and 2 with 0.5 or 1.0 mg / kg agalsidase beta alone (period 1) or in combination with 150 mg migalastat (period 2 )

In FIG. Figure 2 shows the increase in plasma AUC activity of α-Gal A for all patients after co-administration with migalastat.

In FIG. Figure 3 shows partial AUCs for each sampling time, which showed increased plasma α-Gal A activity when co-administered with 0.5 or 1.0 mg / kg agalsidase beta and 150 mg migalastat.

In FIG. 4A-B shows an increase in α-Gal A activity in the skin in two patients after co-administration of 0.5 mg / kg agalsidase beta and 150 mg migalastat.

In FIG. 5A-B shows an increase in α-Gal A activity in the skin in two patients after co-administration of 0.5 mg / kg agalsidase beta and 150 mg migalastat. In FIG. 5A shows an increase in αGal A activity in the skin after co-administration of 0.5 mg / kg of agalsidase beta and 150 mg of migalastat in a patient who received a longer ERT infusion by 40 min for a period of 2.

In FIG. 6A-B shows an increase in α-Gal A activity in the skin in two patients after co-administration of 1.0 mg / kg agalsidase beta and 150 mg migalastat.

In FIG. 7A-B shows an increase in α-Gal A activity in PBMCs in two patients after co-administration of 0.5 mg / kg agalsidase beta and 150 mg migalastat.

In FIG. 8A-B shows an increase in α-Gal A activity in PBMC in two patients after co-administration of 0.5 mg / kg agalsidase beta and 150 mg migalastat.

In FIG. 9A-B shows an increase in α-Gal A activity in PBMCs in two patients after co-administration of 1.0 mg / kg agalsidase beta and 150 mg migalastat.

In FIG. Figure 10 shows a summary table of the increase in α-Gal A activity in plasma, skin, and PBMC after co-administration of 0.5 or 1.0 mg / kg of agalsidase beta and 150 mg of migalastat.

In FIG. 11 shows the genotype of each test subject from examples 2 and 3.

In FIG. 12 shows AUC of α-Gal A plasma activity depending on treatment with 1.0

- 2 031874 mg / kg agalsidase beta and treatment with a combination of 1.0 mg / kg agalsidase beta and 150 mg migalastat

HCl (with averages and standard deviations included).

In FIG. Figure 13 shows the plasma AUC of α-Gal A activity depending on treatment with 0.5 mg / kg agalsidase beta and treatment with a combination of 0.5 mg / kg agalsidase beta and 150 mg migalastat HCl (with mean values and standard deviations included) )

In FIG. 14 shows the activity of α-Gal A in the skin on day 2 after treatment with agalsidase beta alone or in combination with 150 mg migalastat HCl (with coefficients obtained by subtracting the initial value for agalsidase beta separately).

In FIG. 15 shows the activity of α-Gal A in the skin on day 7 after treatment with agalsidase beta alone or in combination with 150 mg of migalastat HCl (with coefficients obtained by subtracting the initial value for agalsidase beta separately).

Detailed description

The present application provides a dosage regimen and administration schedule for the use of 1-deoxyhalactonojirimycin and agalsidase for the treatment of Fabry disease.

Definitions

Fabry disease refers to classic Fabry disease, late-onset Fabry disease, and hemizygous women with mutations in the gene encoding α-galactosidase A (α-Gal A). The term Fabry disease, as used herein, further includes any condition in which a subject exhibits an activity of endogenous α-Gal A below normal.

The term AUC is a mathematical calculation for evaluating the total effect of a given drug over time on an organism. In the graph representing the concentration in the blood after the dose, the concentration variable of the drug is on the y axis, and time is on the x axis. The area between the drug concentration curve and the x axis for a given time interval is AUC. AUCs are used as a guide for dosing regimens and to compare the availability of various drugs in the body.

The term Cmax represents the maximum plasma concentration achieved after dosing.

The terms therapeutically effective dose and effective amount refer to the amount of a particular pharmaceutical compound or composition that is sufficient to provide a useful therapeutic response. A useful therapeutic response can be any response that the user recognizes (for example, a clinician) as an effective response to therapy, including the aforementioned symptoms and surrogate clinical markers. Thus, the therapeutic response will usually be the alleviation of one or more symptoms of a disease or disorder, for example, Fabry disease, such as those known in the art for a disease or disorder, for example, Fabry disease.

Non-limiting examples of improvements in surrogate markers of Fabry disease include increased levels of α-GAL or activity in cells (eg, fibroblasts) and tissues; reduction in GL-3 accumulation, measured by changes in interstitial capillary kidney biopsies using histology; decreased urinary GL-3 levels; assessment of renal function (including glomerular filtration rate (GFR) and protein in the urine in 24 hours); reduced plasma concentrations of homocysteine and vascular endothelial type 1 adhesion molecules (VCAM-1); reduced accumulation of GL-3 in myocardial cells and valve fibrocytes; reduction of heart hypertrophy (especially of the left ventricle), weakening of valve insufficiency and arrhythmias; weakening of proteinuria; decreased urinary lipid concentrations such as STN, lactosylceramide, ceramide, and increased urinary glucosylceramide and sphingomyelin concentrations (Fuller et al., Clinical Chemistry. 2005; 51: 688-694); the absence of layered inclusion bodies (zebra bodies) in the glomerular epithelial cells; improve renal function; weakening of hypohidrosis; the absence of angiokeratitis and the weakening of hearing impairment, such as high-frequency sensorineural hearing loss, progressive hearing loss, sudden deafness or ringing in the ears. Mitigating neurological symptoms include the prevention of transient cerebrovascular accident (TIA) or stroke, and the reduction of neuropathic pain, manifested as acroparesthesia (burning sensation or tingling in the extremities).

The phrase pharmaceutically acceptable refers to molecular units and compositions that are physiologically tolerable and usually do not lead to adverse reactions when administered to humans. Preferably, the term “pharmaceutically acceptable” as used herein means approved by the regulatory authority of the federal or state government or listed on the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans. The term carrier refers to a diluent, excipient, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils. As carriers, in particular for injectable solutions, it is preferable to use water or aqueous saline solutions and aqueous solutions of dextrose and glycerol. Suitable pharmaceutical carriers are disclosed in Remington's Pharmaceutical Sciences, E.W. Martin, 18th Edition or other publications that are incorporated herein by reference in their entirety.

1-deoxygalactone-jirimycin (DGJ) refers to (2R, 3S, 4R, 5S) -2- (hydroxymethyl) piperidine 3,4,5-triol. As used herein, reference to 1-deoxyhalactone-jirimycin or DGJ universally includes both the free base and any form of pharmaceutically acceptable salts of the same. The hydrochloride salt of DGJ is known as migalastat hydrochloride.

The term adjuvant or adjuvant therapy refers to any additional substance, treatment or procedure used to increase the effectiveness, safety or otherwise promote or improve the characteristics of the primary substance, method of treatment or procedure.

The term combined therapeutic agent refers to any therapeutic agent, where the results are improved compared with the effect of each therapeutic agent administered separately. Individual therapeutic agents in a combination therapeutic agent may be administered simultaneously or sequentially.

Strengthening may include any improvement in the effect of various therapies, which may lead to a favorable result compared with the results achieved with therapies carried out separately. The enhanced effect and the determination of the enhanced effect can be measured using various parameters, such as, but not limited to: time parameters (for example, treatment duration, recovery time, long-term treatment effect or treatment reversibility); biological parameters (e.g., cell number, cell volume, cell structure, tissue volume, tissue size, tissue structure); spatial parameters (e.g., tissue strength, tissue size, or tissue accessibility) and physiological parameters (e.g., changes in body shape, pain, discomfort, recovery time, or visible signs). The enhanced effect may include synergistic enhancement, where the enhanced effect is superior to the additive effects of each therapy administered separately. The enhanced effect may include additive enhancement, where the enhanced effect is essentially equal to the additive effect of each therapy carried out separately. The enhanced effect may include less synergistic effect, where the enhanced effect is lower than the additive effect of each therapy carried out separately, but still better than the effect of each therapy carried out separately.

The terms approximately and approximately, in general, should mean an acceptable degree of deviation for the measurand taking into account the nature or accuracy of the measurements. Typically, exemplary degrees of deviation are within 20%, preferably within 10%, and more preferably within 5% of a predetermined value or range of values. Alternatively, and in particular in biological systems, the terms approximately and approximately can mean values that are within the order of magnitude, preferably within 5 times, and more preferably within 2 times of the given value. The numerical values given in this document are approximate unless otherwise indicated.

Composition and introduction.

1-Deoxygalactone-jirimycin can be administered in the form of a free base or in the form of a pharmacologically acceptable salt, including 1-deoxygalactone-jirimycin hydrochloride (otherwise called migalastat hydrochloride). It can be administered in a form suitable for any route of administration, including, for example, orally in the form of tablets, capsules or liquid, or in the form of a sterile injectable aqueous solution. It can be administered orally in the form of tablets, capsules, vaginal ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes or dry powder for mixing with water or another suitable vehicle before use, optionally with flavoring and coloring agents, for use with immediate, delayed, altered, prolonged, intermittent or controlled release. Solid compositions such as tablets, capsules, lozenges, lozenges, dragees, boluses, powders, pastes, granules, pills or preparations of pre-prepared mixtures can also be used. Solid and liquid compositions for oral administration can be obtained in accordance with methods well known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients, which may be in solid or liquid form. If the compound is formulated for oral administration, tablets or capsules can be prepared by conventional methods with pharmaceutically acceptable excipients, such as binders (for example, pregelatinized starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydroorthophosphate); lubricants (e.g. magnesium stearate, talc or silicon dioxide); baking powder (e.g. potato starch or sodium starch glycolate); or moisturizers (e.g. sodium lauryl sulfate). Tablets can be coated using methods well known in the art.

Pharmaceutically acceptable excipients also include, but are not limited to, microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex granulating binders such as polyvinylpyrrolidone, hydroxypropylethyl cellulose (HPMC), hydroxypropyl cellulose (LDC), sucrose, gelatin and gum arabic. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

In a specific embodiment, a mixture of migalastate hydrochloride, magnesium stearate and pregelatinized starch in a white hard gelatin capsule is made up. In another embodiment, the solid dosage form contains about 75-80% migalastate hydrochloride, about 0.1-2% magnesium stearate, and about 20-25% pregelatinized starch. In another specific embodiment, the capsule contains about 76.5% migalastat hydrochloride, about 0.5% magnesium stearate, and about 23% pregelatinized starch.

Enzyme replacement therapy.

Currently approved treatment for Fabry disease is enzyme replacement therapy. Two products are currently available for treating Fabry disease: agalsidase alpha (Replagal®, Shire Human Genetic Therapies) and agalsidase beta (Fabrazyme®; Genzyme Corporation), which are sold worldwide. These two forms of ERT are designed to compensate for a patient's lack of a-Gal A activity using a recombinant form of the enzyme administered intravenously. ERT has been demonstrated to reduce GL-3 accumulation in the endothelium of the kidney capillaries and certain other cell types. Although ERT is effective in many situations, treatment also has limitations. ERT has not shown a reduced risk of stroke, the heart muscle responds slowly, and the elimination of GL-3 from some types of kidney cells is limited. Some patients develop an immune response to ERT.

The recommended dosage of agalsidase alpha is 0.2 mg / kg body weight, which is administered by infusion every 2 weeks as an intravenous infusion. A 10-week study was conducted in adult patients with Fabry disease not previously exposed to ERT to evaluate the pharmacokinetics and pharmacodynamics of agalsidase alpha. The average elimination half-life after administration of doses in the range from 0.1 to 0.4 mg / kg of agalsidase alpha was 56-76 min without a significant relationship between the dose and elimination half-life, clearance or volume of distribution. AUC was a linearly proportional dose in this dose range. Plasma GL-3 levels decreased in groups with all doses by approximately 50%; this decrease was independent of dose and dosing frequency. During the study, two of the 18 patients became IgG positive. During the study, no patients found IgE antibodies.

The recommended dosage of agalsidase beta is 1 mg / kg body weight, which is administered every 2 weeks as an intravenous infusion. The manufacturer of agalsidase beta reports a drug shortage, while in the case of Fabry disease in the USA, only ERT is approved. As a consequence, agalsidase beta is currently normalized, and patients usually receive a reduced dose of the enzyme and / or have an extended dosing interval (i.e. more than 2 weeks between doses). Agalsidase beta shows non-linear pharmacokinetics with increasing exposure values (AUC) and decreasing clearance disproportionately with increasing doses. AUC values increased approximately 6 times and 8 times with increasing doses from 0.3 to 1 mg / kg and from 1 to 3 mg / kg, respectively. The half-life of agalsidase beta in adult patients after application of doses varying from 0.3 to 3 mg / kg depended on the dose and varied from 45 to 100 minutes.

IgG antibodies to agalsidase beta appeared in 79% of adult patients and 69% of pediatric patients treated with agalsidase in clinical trials; however, in most patients who developed IgG antibodies, this occurred during the first 3 months of exposure. Male patients, especially those with low residual levels of α-Gal A, were more likely to develop IgG antibodies than male patients with higher residual levels or female patients. IgG seroconversion in pediatric patients has been associated with a prolonged half-life of agalsidase. However, in adult patients, identical pharmacokinetic agalsidase profiles were observed before and after seroconversion in one trial; in another trial, maximum agalsidase concentrations and AUC values were reduced to 26% of baseline in patients with the highest IgG titers. The presence of IgG antibodies to agalsidase has been reported to reduce enzyme activity.

Migalastat hydrochloride stabilizes in vitro α-Gal A wild-type, as well as in vivo. In vitro, the binding of migalastat hydrochloride to rha-Gal A has been shown to lead to a significant time and concentration-dependent increase in the stabilization of rha-Gal A at neutral pH, as measured by thermal denaturation and activity. In a pH neutral buffer, rha-Gal A showed a loss of activity with a half-life of approximately 3 hours; co-incubation with migalastat hydrochloride increased the elimination half-life due to the loss of rha-Gal A activity to approximately 40 hours

In rats, oral administration of 3 mg / kg of migalastat hydrochloride followed by injection after 30 min of 10 mg / kg of agalsidase beta led to a 2.6-fold increase in the half-life of rha-Gal A from plasma and a 2.5- and 1.5-fold increase plasma a-Gal A levels after 60 and 240 min, respectively. In mice with GLA deficiency, oral administration of doses of 30, 100 or 300 mg / kg of migalastat hydrochloride 5301874 in the 30 minutes before and 2 hours after rha-Gal A injection led to a dose-dependent increase in tissue levels of α-Gal A and a dose-dependent decrease levels of GL-3 in the skin, heart, kidneys and plasma compared with the administration of rha-Gal A alone.

Migalastat hydrochloride has been shown to stabilize agalsidase alfa both in vitro and in vivo. The effect of migalastat hydrochloride on the physical stability of alpha agalsidase was evaluated using an in vitro thermal denaturation assay. Using this assay, agalsidase alpha showed a melting point (Tm) of approximately 51 ° C at pH 7.4. However, when 10 μM migalastat hydrochloride Tm agalsidase alpha was included in the denaturation reaction, it increased significantly to 59 ° C. As expected for the lysosomal enzyme, agalsidase alpha was more stable at low pH (Tm 58 ° C at pH 5.2) and showed additional resistance to thermal denaturation in the presence of 10 μM migalastat hydrochloride (Tm 68 ° C). These data show that the binding of migalastat hydrochloride gives agalsidase alpha a high level of physical stability.

The effect of migalastat hydrochloride on the clearance rate of agalsidase alpha from the blood of male Sprague-Dawley rats was also investigated. Animals received a vehicle (water) or a single oral administration through a probe of 1, 3, 10 or 30 mg / kg of migalastat hydrochloride, and then after 30 minutes intravenous administration of 0.2 mg / kg of agalsidase alpha by intravenous bolus injection into the tail vein. Blood was collected versus time and plasma α-Gal A activity was measured. In the absence of migalastat hydrochloride, α-Gal A activity rapidly decreased; preliminary administration of migalastat hydrochloride resulted in a dose-dependent increase in the half-life of agalsidase alpha (as measured by α-Gal A activity) by approximately 2 times and 3 times after administration of 3 and 30 mg / kg migalastat hydrochloride, respectively, with approximately 2.5 and 1.5 -fold increase in plasma levels of α-Gal A after 60 and 240 minutes, respectively. The effect of migalastat hydrochloride on agalsidase alpha, both in vitro and in vivo, is comparable to the observed effect of migalastat hydrochloride on agalsidase beta.

A preliminary study in GLA-deficient mice was performed with a safety assessment of the co-administered migalastat hydrochloride and Fabrazyme®. Migalastat hydrochloride was administered three times a week for four weeks at doses of 3 and 30 mg / kg in combination with Fabrazyme®, administered intravenously once a week at a dose of 1 mg / kg. Apparently, there are no direct drug-related changes in survival, clinical status or hematology and clinical chemistry observed in male GLA-deficient mice co-administered with migalastat hydrochloride and Fabrazyme®.

Examples

Example 1. The dosage regimen for the treatment of Fabry disease using migalastat hydrochloride and agalsidase.

One objective of the study is to evaluate the safety, efficacy, and pharmacodynamics of dosing regimens, including co-administration of migalastat hydrochloride and agalsidase in patients with Fabry disease.

Another objective of the study is to evaluate the effects of doses of migalastat hydrochloride at 150 and 450 mg on the distribution of α-Gal A. It will be evaluated by measuring the distribution of agalsidase in the skin after administration of a dose of agalsidase separately and agalsidase in combination with migalastat hydrochloride 24 hours and 7 days after administration doses by measuring α-Gal A levels and protein levels.

Other dimensions to be evaluated are:

the allocation of GL-3 with urine before each dose of agalsidase and 14 days after it;

GL-3 in the skin after administration of a dose of agalsidase separately and agalsidase in combination with migalastat hydrochloride 24 hours and 7 days after the dose;

α-Gal A enzyme levels in WBC, determined before the start of the agalsidase infusion and after 2, 4 and 24 hours, and 7 and 14 days after the dose;

antibody titer (IgG) before the start of the agalsidase infusion;

plasma concentrations of globotriaosylsphingosine (lyso-GB3) and urinary lyso-GB3 excretion before each dose of agalsidase and 14 days after each dose of agalsidase.

All measurements of the levels of α-Gal A enzyme in plasma, WBC, and skin are carried out with and without capture of concanavalin A (Con A), and protein levels are determined by Western blotting.

Study design.

This is a phase 2 clinical, two-stage, open-label study to evaluate the safety and effectiveness of co-administration of migalastat hydrochloride and agalsidase. The study will be conducted on male subjects from 18 to 65 years of age who received stable doses (0.3-1.0 mg / kg) of agalsidase beta (Fabrazyme®) or (> 0.2 mg / kg) of agalsidase alpha (Replagal®) at least one month before the start of the study. Approximately 18 subjects will be included.

This open research will consist of two stages. Stage 1 will consist of a screening and a three-period study to evaluate the effect of 150 mg of migalastat hydrochloride on the pharmacokinetics and safety of agalsidase and the effect of agalsidase on the pharmacokinetics and safety of 150 mg migalastat hydrochloride. Stage 2 will consist of a screening and two-period study to evaluate the effect of 450 mg migalastat hydrochloride on the pharmacokinetics and safety of agalsidase. At stage 2, the effect of agalsidase on the pharmacokinetics and safety of a dose of 450 mg migalastat hydrochloride will not be evaluated. The content of migalastat hydrochloride in plasma with the introduction of migalastat hydrochloride with agal sidase will be determined solely to confirm the achievement of adequate concentrations of migalastat hydrochloride in the plasma.

Each subject will receive each of the following treatments in the order described below. Stage 1 will consist of the following periods:

Period 1: agalsidase alone as an intravenous infusion.

Period 2: an oral dose of 150 mg of migalastat hydrochloride two hours before the start of the intravenous infusion of agalsidase.

Period 3: oral dose of 150 mg of migalastat hydrochloride. The dose of agalsidase administered in periods 1 and 2 will be identical. Agalsidase alpha will be administered as a 40-minute intravenous infusion, and agalsidase beta will be administered as a 2-hour intravenous infusion.

As for period 1, before the next planned infusion of agalsidase in subjects, the following assessments will be made: assessment of side effects, taking additional medications, medical examination, weight measurement, vital signs, 12-lead ECG, clinical laboratory tests (serum biochemical analysis, hematological analysis and general analysis of urine), skin biopsy (puncture biopsy to measure the levels of the enzyme α-Gal A; if an appropriate sample is available, GL-3 in the skin will also be determined e).

On the morning of day 1, urine will be collected to determine GL-3 and lyso-GB3 in the urine, and then the current dose of agalsidase for the subject will be administered, which is given as an infusion using an infusion pump. Blood samples for pharmacokinetic and pharmacodynamic analysis will be taken immediately before the start of the agalsidase infusion and within a 24-hour period after the start of the agalsidase infusion. Plasma α-Gal A enzyme levels and WBC, plasma lyso-GB3 and plasma antibody titer will be determined in the collected blood samples for the time periods given in table. 2 for agalsidase beta and in table. 4 for agalsidase alpha. An 12-lead ECG will be performed at the end of the agalsidase infusion, immediately after sampling after an infusion blood sample.

On day 2, 24 hours after the start of the infusion of the previous day, a puncture biopsy of the skin will be taken, in which the levels of the enzyme α-Gal A will be determined; if an appropriate sample is available, GL-3 in the skin will also be detected. After taking the last pharmacokinetic test, the following assessments will be made: the assessment of side effects, the use of additional drugs, medical examination, weight measurement, vital signs, as well as clinical laboratory tests (serum biochemical analysis, hematological analysis and general urine analysis).

On day 7, the subjects will make the following assessments: a medical examination, an assessment of the basic physiological parameters, taking additional medications and an assessment of side effects. A skin biopsy will be taken in which the levels of the α-Gal A enzyme are determined; if an appropriate sample is available, GL-3 in the skin will also be detected. A blood sample will also be taken to determine plasma levels of α-Gal A enzyme and WBC. On day 14, a urine sample will be taken to determine the excretion of GL-3 and lyso-GB3 in the urine. A blood sample will also be taken to determine the levels of the α-Gal A enzyme in plasma and WBC, and vital signs will be evaluated.

As for period 2, before the next planned infusion of agalsidase, the subjects will make the following assessments: assessment of side effects, taking additional medications, medical examination, weight measurement, vital signs, 12-lead ECG, clinical laboratory tests (serum biochemical analysis, hematological analysis and general analysis of urine).

In the morning on day 1, urine will be collected to determine GL-3 and lyso-GB3 in the urine, and then an oral dose of 150 mg of migalastat hydrochloride will be administered 2 hours before the planned infusion of agalsidase. Subjects will refrain from eating at least 2 hours before and 2 hours after the administration of migalastat hydrochloride. In period 2, each subject will receive a dose of agalsidase identical to that administered in period 1, as an infusion using an infusion pump. Agalsidase infusion will begin 2 hours after a dose of migalastat hydrochloride is administered.

Blood samples for pharmacokinetic and pharmacodynamic analysis will be taken before the dose of migalastat hydrochloride is administered and 1 hour after the administration of migalastat hydrochloride. Additional blood samples will be taken immediately before the start of the agalsidase infusion and within a 24-hour period after the start of the agalsidase infusion. Plasma α-Gal A enzyme levels and WBC, plasma lyso-GB3, and plasma antibody titer will be determined in collected blood samples for the time periods given in Table 1. 2 for agalsidase beta and in table. 4 for agalsidase alpha. An ECG in 12 leads will be performed at the end of the agalsidase infusion, immediately after sampling after blood infusion samples.

On day 2, 24 hours after the start of the infusion of the previous day, a puncture biopsy of the skin will be taken, in which the levels of the enzyme α-Gal A will be determined; if an appropriate sample is available, GL-3 in the skin will also be detected. After taking the last pharmacokinetic test, the following

- 7 031874 evaluations: evaluation of side effects, taking additional medicines, medical examination, weight measurement, vital signs, as well as clinical laboratory tests (serum biochemical analysis, hematological analysis and general urine analysis).

On day 7, the subjects will make the following assessments: a medical examination, an assessment of the basic physiological parameters, taking additional medications and an assessment of side effects. A skin biopsy will be taken, in which the levels of the enzyme α-Gal A will be determined; if an appropriate sample is available, GL-3 in the skin will also be detected. A blood sample will also be taken to determine plasma levels of α-Gal A enzyme and WBC.

On day 14, a urine sample will be taken to determine the excretion of GL-3 and lyso-GB3 in the urine. A blood sample will also be taken to determine the levels of the α-Gal A enzyme in plasma and WBC, and vital signs will be evaluated.

After all evaluations are completed after period 2, subjects begin period 3. All subjects will receive the next agalsidase infusion on day 1 based on their usual dosing schedule. On day 6, all subjects will perform the following evaluations: assessment of side effects, taking additional medications, medical examination, weight measurement, vital signs, 12-lead ECG, as well as clinical laboratory tests (serum biochemical analysis, hematological analysis and general analysis urine).

On day 7, an oral dose of 150 mg of migalastat hydrochloride will be administered. Subjects will refrain from eating at least 2 hours before and 2 hours after the administration of migalastat hydrochloride. Blood samples will be taken before the dose and within a 24-hour period after the introduction of migalastat hydrochloride. The concentrations of migalastat hydrochloride are measured in all plasma samples (see the sampling times in Table 2 for agalsidase beta and in Table 4 for subjects receiving agalsidase alpha).

On day 8 after the last pharmacokinetic sample is taken, the following assessments will be made: an assessment of side effects, taking additional medications, a medical examination, basic vital functions, as well as clinical laboratory tests (serum biochemical analysis, hematological analysis and general urine analysis).

Surveillance after period 3 will be carried out by telephone 28 days after period 3. The following evaluations will be made: taking additional drugs and side effects.

As for stage 2, each subject will receive each of the following treatments in the order described below:

Period 1: agalsidase alone as an infusion.

Period 2: an oral dose of 450 mg of migalastat hydrochloride two hours before the start of the intravenous infusion of agalsidase.

The dose of agalsidase administered in periods 1 and 2 will be identical. Agalsidase alpha will be administered as a 40-minute intravenous infusion, and agalsidase beta will be administered as a 2-hour intravenous infusion.

As for period 1, for subjects meeting all selection criteria, the following evaluations will be performed before the next planned infusion of agalsidase: assessment of side effects, additional medication, medical examination, weight measurement, vital signs, 12 lead ECG, clinical laboratory tests (serum biochemical analysis, hematological analysis and general urine analysis), skin biopsy (puncture biopsy to measure α-Gal A enzyme levels; if appropriate th sample, and determine the GL-3 in the skin).

On the morning of day 1, urine will be collected to determine GL-3 and lyso-GB3 in the urine, and then the current dose of agalsidase for the subject will be administered, which is given as an infusion using an infusion pump. Blood samples for pharmacokinetic and pharmacodynamic analysis will be taken immediately before the start of the agalsidase infusion and within a 24-hour period after the start of the agalsidase infusion. Plasma α-Gal A enzyme levels and WBC, plasma lyso-GB3, and plasma antibody titer will be determined in collected blood samples for the time periods given in Table 1. 3 for agalsidase beta and in table. 5 for agalsidase alpha. An 12-lead ECG will be performed at the end of the agalsidase infusion, immediately after sampling after an infusion blood sample.

On day 2, 24 hours after the start of the infusion of the previous day, a puncture biopsy of the skin will be taken, in which the levels of the enzyme α-Gal A will be determined; if an appropriate sample is available, GL-3 in the skin will also be detected. After taking the last pharmacokinetic test, the following assessments will be made: the assessment of side effects, the use of additional drugs, medical examination, weight measurement, vital signs, as well as clinical laboratory tests (serum biochemical analysis, hematological analysis and general urine analysis).

On day 7, subjects will perform the following assessments: vital signs, supplementation, and side effects. A biopsy of the skin in which

- 8 031874 determine the levels of the enzyme α-Gal A; if an appropriate sample is available, they will also determine

GL-3 in the skin. A blood sample will also be taken to determine the plasma level of α-Gal A enzyme and WBC.

On day 14, urine samples will be taken to determine the excretion of GL-3 and lyso-GB3 in the urine.

A blood sample will also be taken to determine the levels of the α-Gal A enzyme in plasma and WBC, and vital signs will be evaluated.

As for period 2, before the next planned infusion of agalsidase, the subjects will make the following assessments: assessment of side effects, taking additional medications, medical examination, weight measurement, vital signs, 12-lead ECG, clinical laboratory tests (serum biochemical analysis, hematological analysis and general analysis of urine).

On the morning of day 1, urine will be collected to determine GL-3 and lyso-GB3 in the urine, and then an oral dose of 450 mg of migalastat hydrochloride will be administered 2 hours before the planned infusion of agalsidase. Subjects will refrain from eating at least 2 hours before and 2 hours after the administration of migalastat hydrochloride. In period 2, each subject will receive a dose of agalsidase identical to that administered in period 1, as an infusion using an infusion pump. Agalsidase infusion will begin 2 hours after a dose of migalastat hydrochloride is administered.

Blood samples for pharmacokinetic and pharmacodynamic analysis will be taken before the dose of migalastat hydrochloride is administered and 1 hour after the administration of migalastat hydrochloride. Additional blood samples will be taken immediately before the start of the agalsidase infusion and within a 24-hour period after the start of the agalsidase infusion. Plasma α-Gal A enzyme levels and WBC, plasma lyso-GB3, and plasma antibody titer will be determined in collected blood samples for the time periods given in Table 1. 3 for agalsidase beta and in table. 5 for agalsidase alpha. An 12-lead ECG will be performed at the end of the agalsidase infusion, immediately after sampling after an infusion blood sample.

On day 2, 24 hours after the start of the infusion of the previous day, a skin biopsy will be taken, in which the levels of the enzyme α-Gal A will be determined; if an appropriate sample is available, GL-3 in the skin will also be detected. After taking the last pharmacokinetic test, the following assessments will be made: the assessment of side effects, the use of additional drugs, medical examination, weight measurement, vital signs, as well as clinical laboratory tests (serum biochemical analysis, hematological analysis and general urine analysis).

On day 7, subjects will perform the following assessments: vital signs, supplementation, and side effects. A skin biopsy will be taken to determine the levels of the α-Gal A enzyme. A blood sample will also be taken to measure the plasma levels of α-Gal A enzyme and WBC.

On day 14, urine samples will be taken to determine the excretion of GL-3 and lyso-GB3 in the urine. A blood sample will also be taken to determine the level of α-Gal A enzyme in plasma and WBC, and vital signs will be evaluated.

The follow-up will be 28 days after period 2. The following assessments will be made: taking additional drugs and side effects.

Assessment and sampling schemes.

In the table. Figure 1 shows the evaluation scheme in steps 1 and 2. The sampling time and the analyzed samples for co-administration of migalastat hydrochloride with Fabrazyme® are given in table. 2 and 3. Sampling times and test samples for co-administration of migalastat hydrochloride with Replagal® are given in table. 4 and 5.

- 9 031874

Table 1

Grade scheme (steps 1 and 2)

Act Informed consent Medical history and demographic data Screening Within 28 days after screening X X Pe Day 1 Rio 1 and Day 1 2 (this Day 2 you are 11 day 7 2) Day 14 Per | Day 1 iodine 3 (this g Day 6 only ia 1) Day 7 'FOR Day 8 Follow-up (steps 1 and 2) Telephone contact Medical examination X X X X X X ECG (12 leads) X X X X X Vital signs ⁶ X X X X X X X X X Hematological analysis X X X X X General urine analysis X X X X X Biochemical analysis of serum X X X X X Additional Drug Information X X X X X X X X X X eGFR X GL-3 / LysoGB3 in urine X X Registration procedure X X Dose of drug X ¹ X ² X ³ RK blood sampling ⁴ X X X X X X Skin Biopsy ⁴ x ⁶ X X WBC selection X X X X Antibody titer ⁴ X Side effects X X X X X X X X X

¹ - administered drugs: agalsidase (periods 1 and 2), migalastat G ^ period 2);

² - outpatient administration of Fabrazyme® or Replagal®;

³ - administered drug: migalastat HCl;

⁴ - sampling time and analyzed samples are given in table. 2 and 3 for Fabrazyme® (agalsidase beta) and tab. 4 and 5 for Replagal® (agalsidase alpha);

⁵ - only for period 1;

⁶ - vital signs include temperature, blood pressure, heart rate, and respiration.

table 2

FABRAZYME (agalsidase beta) - stage 1: selection of blood, urine and skin.

Moments and analysis of samples.

Period 1 Screening Scheme ___________ After the start of the infusion Fabrazyme ® (h) ___________ Day

Sampling 0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 12 24 7 14

Blood X X X X X X X X X X X X X X X X Leather X X X Urine X X

Fabrazyme® Infusion

^Plume Sample ^Analysis | ^ 'I ^x ! ^Χ ΓΙΊ ^Χ Ι ^Χ Ι ^x ! ^Χ ΓΙ ^Χ ΓΙΊ ^Χ Ι ^X 114 ^x

- 10 031874

Antibody X α-Gal A in Wbc X X X X X X Lyso-gb3 X X X Leather α-Gal A ’ X X X Urine GL-3 X X Lyso-gb3 X X

Period 2 After flashing astatine on Selection scheme After the start of infusion Fabrazyme ® (h) Day Sample taken 0 1 0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 10 12 24 7 14

Blood x | x X X X X X X X X X X X X X X X Leather X Urine X 1

The introduction of migalastat X HCl

Fabrazyme ® Infusion (arrow)

After a dose of migalastat HCI

Sampling 01 2 3 4 5 6 7 810 12 24

Blood X X X X X X X X X X X X Leather Urine

The introduction of migalastat HCI

Analysis p rob | Plasma Migalastat | _{x x x x x} X X X X X X X

If an appropriate sample is available, GL-3 in the skin will also be measured.

Table 3

FABRAZYME (agalsidase beta) - stage 2 - selection of blood, urine and skin. Moments and analysis of samples.

Selection scheme

Period 1 ___________ After the start of the infusion Fabrazyme ® (h) __________

Sampling 0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 12 24

Blood X X X X X X X X X X X X X X Leather X X Urine X

Fabrazyme® Infusion

Sample Analysis

Plasma b-gal a c plasma X X X X X X X X X X X X X X Antibody X b-gal a c Wbc X X X X Lyso-gb3 X Leather b-Gal A ’ X X Urine GL-3 X Lyso-gb3 X

Period 2

After migalastat HCI

Selection scheme

After the start of infusion Fabrazyme ® (h)

0.5 1 1.5 2 2.5 3 4 5 6 7 8 10 12 24

Day

14

Selected θ

Blood X X X X X X x | x X X X X X X X X X Leather X Urine X

The introduction of migalastat X HCI

Infusion

Fabrazyme ® (arrow)

Sample Analysis

Plasma b-Gal A in plasma X X X X X X X X X X X X X X X Blink Asta that BUT X X X X X X X X X X X X Antibody X b-Gal A in WBC X X X X Lyso-gb3 X Leather b-Gal A ' X Urine GL-3 X Lyso-gb3 X

If an appropriate sample is available, GL-3 in the skin will also be measured.

- 11 031874

Table 4

REPLAGAL (agalsidase alpha) - stage 1: blood, urine and skin sampling. Moments and analysis of samples.

Selection scheme

Period 1

After the start of Replagal® infusion (h)

Day

12 24

Selected _Q πηηηα ''

I blood X x | x | x | x | x | x | x | x | x X X 1 X 1 X 1 1 Leather X  Ί 1 J 1 1 x 1

I Urine Infusion Replagal®

Sample Analysis

Plasma b-gal a c plasma X X X X X X X X X X X X X X Antibody X b-gal a c Wbc X X X X Lyso-gb3 X Leather b-Gal A * X X Urine GL-3 X Lyso-gb3 X

Period 2

Selection scheme

After the start of Replagal® infusion (h)

6 7 8 10 12 24

Day

0.33 0.66 1 1.5

After migalastat HC1 θ sample taken

Blood X X X X X X X X X X X X X X X X X Leather X Urine X

Introduction of migalast and HC1 Replagal® infusion (arrow)

Sample Analysis

Plasma b-gal a c plasma X X X X X X X X X X X X X X X Migalastata HC1 X X X X X X X X X X X X Antibody X b-gal a c Wbc X X X X Lyso-gb3 X Leather b-Gal A ’ X Urine GL-3 X Lyso-gb3 X

Period 3

Sampling scheme)

After a dose of migalastat HC1 ___________

10 12 24

Blood X X X X X X X X X X X X Leather Urine

Sample Analysis

Introduction migalastat HCl X | Plasma | Migalastat HC1 | x | x | x | x | x | x | x | x | x | x | x [x |

If an appropriate sample is available, GL-3 in the skin is also measured.

- 12 031874

4 5

Table 5

REPLAGAL (agalsidase alpha) - stage 2: blood, urine and skin sampling. Moments and analysis of samples.

Period 1 Selection Scheme ________________ After the start of Replagal® infusion (h) _______________

The selected 1 1 S 2 3 4 S 6 7 8 12 24 sample, ,’’

Blood X X X X X X X X X X X X X X Leather X X Urine X -

Replagal®

Analysis w

Plasma b-gal a c plasma X X X X X X X X X X X X X X Antibody X b-gal a c Wbc X X X X Lyso-gb3 X Leather b-Gal A ’ X X Urine GL-3 X Lyso-gb3 X

Period 2

Selection scheme

Sample taken

After migalastat

HC1 ________ After the start of the infusion Replagal® (h) ________

1 0 0.33 0.66 1 1.5 2 3 4 5 6 7 8 10 12 24

Blood X X X X X X X X X X X X X X X X X Leather X Urine X

The introduction of migalastat X

HC1

Infusion

Fabrazyme® (arrow)

Days

Sample Analysis

Plasma b-gal a c plasma X X X X X X X X X X X X X X X Migalastata HC1 X X X X X X X X X X X X Antibody X b-gal a c Wbc X X X X Lyso-gb3 X Leather b-Gal A ’ X Urine GL-3 X Lyso-gb3 X

* If an appropriate sample is available, GL-3 in the skin is also measured.

Pharmacokinetics of migalastat HCl and rha-Gal A.

The concentrations of migalastat HCl in blood samples were measured in plasma using a validated LC-MS / MS assay. Plasma α-Gal A levels will be determined using a validated assay measuring enzyme activity using 4-MUG with and without concanavalin A (Con A). Α-Gal A protein levels are measured by Western blotting using a human anti-Gal A antibody.

Α-Gal A enzyme levels in the skin.

Α-Gal A enzyme levels are examined in skin biopsy samples. Skin biopsies will be obtained using a puncture device. At each visit, they will extract one fragment. The levels of α-Gal A in the skin are determined using a validated assay measuring enzyme activity using 4-MUG with and without concanavalinoma A (Con A). Α-Gal A protein levels are measured by Western blotting using a human anti-Gal A antibody.

Α-Gal A levels in WBC.

Levels of α-Gal A in WBC will be determined in blood samples using a validated assay measuring enzyme activity using 4-MUG with and without concanavalin A (Con A). Α-Gal A protein levels are measured by Western blotting using an anti-human Gal A antibody.

Lyso-GB3 in plasma.

Measurement of lyso-GB3 in plasma will be carried out on the basis of an explorative approach to obtain data in patients receiving only ERT and ERT with the joint introduction of migalastat hydrochloride. Concentrations of lyso-GB3 were measured in plasma using a validated assay.

GL-3 and Lyso-GB3 in urine.

The first morning urine sample will be taken from each subject for analysis on the excretion of GL-3 and lysoGB3 with urine on day 1 and day 14 of periods 1 and 2. Subjects collect urine in the morning on days 1 and 14. GL-3 in urine and Lyso-GB3 in urine will be expressed as a function of creatinine concentration in the urine.

Antibody titer.

Blood samples will be taken and IgG antibody titers will be measured in each blood sample.

Security Options

Safety parameters will be evaluated by considering changes in the medical exam data, vital signs, changes in the ECG over time, clinical laboratory tests and side effects.

Indicators of vital functions, weight and height.

Screening and recording will measure body temperature and respiration. To monitor safety, body temperature, respiration, sitting blood pressure, and heart rate will be measured before the dose and approximately 1, 2, 3, 4, and 6 hours after the administration of agalsidase (period 1) or migalastat hydrochloride (periods 2 and 3 ) on days 2, 7 and 14. If the time of monitoring vital signs is the same as blood sampling, blood sampling will have priority, and the monitoring of vital signs will be adjusted accordingly.

ECG monitoring.

ECG monitoring will be performed using a standard 12-lead ECG.

Clinical laboratory studies.

Blood samples for clinical laboratory tests (hematological analysis, serum biochemical analysis) and general urine analysis will be taken at each visit and analyzed in a central laboratory. Hematological studies include studies of total hemoglobin, hematocrit, the number of red blood cells, platelets and white blood cells, and the white blood cell count.

Coagulation (screening only) includes INR and ARTT.

Serum biochemical analysis includes the measurement of AST, ALT, alkaline phosphatase, total bilirubin, creatinine, urea, glucose, calcium, sodium, potassium, magnesium, total protein, albumin, bicarbonate, LDH, blood urea nitrogen, chloride and phosphate. Serum creatinine will be measured using reagents that have been calibrated using the reference isotope dilution mass spectrometry (IDMS) method.

A general urinalysis includes determination of color, appearance, specific gravity, pH, protein, glucose, ketones, blood, leukocyte esterase, nitrites, bilirubin, urobilinogen, as well as microscopy of sediment.

Pharmacokinetic parameters.

The pharmacokinetic parameters AUC _ot, AUC _{is infinite} CHN _{spine, C max, t max, k} cl, calculated nekompartmentnymi methods, and the half-life was calculated from the concentrations migalastata hydrochloride in plasma and the levels of the enzyme α-Gal A. summarize the pharmacokinetic parameters via processing using descriptive statistics. Calculate the ratio of AUC _ot, AUC _{is infinite} CHN _spine for each compound separately to the corresponding compound in combination. Pharmacokinetic and pharmacodynamic data for subjects receiving agalsidase alpha and agalsidase beta are analyzed separately.

Statistical analysis.

Depending on the specific case, descriptive statistics will be provided (N, mean, standard deviation and coefficient of variation, standard error, median, minimum and maximum). The effect of the compound on the jointly administered compound is evaluated by counting the individual (in relation to the subject) AUC and C _max ratios as follows:

The ratio AUC = AUC

AUC infinity! Combination) infinity (separately)

Relation C _max Stax (combination) Stach (separately)

The AUC and C _max ratios will be expressed as the average of the individual relationships with a 90% confidence interval for the average. Pharmacokinetic and pharmacodynamic data for subjects receiving agalsidase alpha and agalsidase beta are analyzed separately. Depending on the specific case, the results will be presented in tabular and graphical form. All subjects who will receive the test drug and have sufficient data to obtain reliable pharmacokinetic parameters are included in the safety analysis and pharmacokinetic analysis.

Example 2. The dosage regimen for the treatment of Fabry disease using migalastat hydrochloride and agalsidase.

Migalastat HCl is a pharmacological chaperone for α-galactosidase A (α-Gal A), which increases the stability and proper folding of the enzyme. Migalastat can act by preventing inactivation of α-Gal A by stabilizing the enzyme at pH / blood temperature. The aim of this study was to study the effects of 150 and 450 mg of migalastat, administered 2 hours before administration of agalsidase, on the safety and pharmacokinetics of plasma agalsidase in subjects with Fabry disease. The aim of this study was also to study the effects of 150 and 450 mg of migalastat on the pharmacokinetics of agalsidase in plasma, skin and PBMC in patients with Fabry disease; study of the effect of plasma agalsidase on the pharmacokinetics of migalastat in plasma; assessment of GL-3 in urine and lyso-GB3 in plasma and urine before and 14 days after agalsidase infusion and assessment of antibody titer before infusion

- 14 031874 agalsidase.

Ways.

The study was carried out in accordance with the methods described in example 1. In particular, it was an open, single dose, non-randomized, fixed sequence 2-stage study.

Stage 1 included 3 periods:

(1) enzyme replacement monotherapy (ERT) of 0.5 or 1.0 mg / kg of agalsidase beta (infusion for about 2 hours) or 0.2 mg / kg of agalsidase alpha (infusion for about 40 minutes);

(2) co-administration of ERT + 150 mg oral migalastat tablets (single dose), with migalastat administered 2 hours before ERT; and (3) monotherapy with 150 mg migalastat.

Stage 2 included 2 periods in the same sequence as stage 1 (i.e., periods (1) and (2)), but with the combined administration of 3x150 mg tablets of migalastat for oral administration in period 2 (i.e. 450 mg migalastat).

Stage 2 included the following 2 periods:

(1) enzyme replacement monotherapy (ERT) of 0.5 or 1.0 mg / kg of agalsidase beta (infusion for about 2 hours) or 0.2 mg / kg of agalsidase alpha (infusion for about 40 minutes); and (2) co-administration of ERT + 450 mg oral migalastat tablets. Migalastat was administered 2 hours before ERT.

Treatment periods were separated by a minimum of a 14-day washout period.

α-Gal A catalyzes the initial step in the cleavage of the GL-3 substrate in vivo. α-Gal A also acts on other substrates with the same α-bond, for example, the artificial low molecular weight 4-MUG fluorescent substrate. The effect of α-Gal A on 4-MUG was measured in vitro in plasma, skin, and PBMC samples after serial dilutions for dissociation of migalastat.

Plasma, skin, and PBMC α-galactosidase A activity were assessed for 6 patients in stage 1, periods 1 and 2. Four patients received 0.5 mg / kg ER agalsidase beta ERT, and 2 patients received 1.0 mg / kg beta agalsidase ERT time of period 1, and co-administration of 150 mg of migalastat 2 hours before the start of the same dose of ERT during period 2. The infusion duration was 2 hours for both periods, with the exception of one: one patient receiving 0.5 mg / kg of agalsidase beta had an unbalanced infusion, where the patient was infused for 2 hours and 40 minutes during the period and 2, but during a period of 1 infusion was performed for only 2 hours.

Results.

Increased plasma α-Gal-A activity with co-administration of migalastat.

Regarding co-administration with migalastat (period 2) with respect to only ERT (period 1), the following average increases in plasma AUC (area under the curve) of α-Gal A activity were observed:

Average increases for infusion of 0.5 mg / kg agalsidase beta:

3.0-fold for 0.5 mg / kg agalsidase beta (N = 4), increase in individual patients: 2.0-, 2.2-, 3.4- and 4.2-fold, average increase, except for the patient with a 2.0-fold increase in case of an unbalanced infusion duration: 3.3-fold.

Average increases for infusion of 1.0 mg / kg agalsidase beta:

1.9-fold for 1.0 mg / kg agalsidase beta (N = 2), increase in individual patients: 1.6-fold and 2.2-fold.

In FIG. 1 shows the combination of α-Gal A activities in the plasma of six patients for periods 1 and

2. Increases in plasma AUC activity of α-Gal A for all patients after co-administration with migalastat are shown in FIG. 2. In FIG. Figure 3 shows partial AUCs for each sampling time, which show increased plasma α-Gal A activity when co-administered with 0.5 or 1.0 mg / kg agalsidase beta with 150 mg migalastat.

Increases in α-Gal-A activity in the skin in the case of migalastat.

Regarding co-administration with migalastat (period 2) with respect to only ERT (period 1), the following average increases in α-Gal A activity in the skin were observed:

Average increases for infusion of 0.5 mg / kg agalsidase beta:

2.6-fold for 0.5 mg / kg agalsidase beta per day 2 (N = 3, 1 patient sample lost), increases in individual patients: 2.8-, 3.9- and 1.1-fold, no changes in activity in the skin of period 1 on day 7.

Average increases for infusion of 1.0 mg / kg agalsidase beta:

1.9- and 1.5-fold for 1.0 mg / kg agalsidase beta on days 2 and 7, respectively (N = 2), increases in individual patients: 1.6- and 2.1-fold on day 2, 1, 7 and 1.2 times a day 7.

An increase in α-Gal A activity in the skin after co-administration of 0.5 or 1.0 mg / kg of agalsidase beta and 150 mg of migalastat is shown in FIG. 4-6. In FIG. 5A shows an increase in α-Gal A activity in the skin after co-administration of 0.5 mg / kg of agalsidase beta and 150 mg of migalastat in a patient who

- 03 031874 time period 2 received an ERT infusion 40 minutes longer than other patients.

Increases in α-Gal-A activity in PBMC in the case of migalastat.

Regarding co-administration with migalastat (period 2) with respect to only ERT (period 1), the following average increases in α-Gal A activity in PBMC were observed:

Average increases for infusion of 0.5 mg / kg agalsidase beta:

2.3-, 2.0- and 2.2-fold for 0.5 mg / kg agalsidase beta (N = 4) on days 2, 7 and 14, respectively, the ranges of increases in individual patients: 1.4-3, 1-, 1.4-2.3- and 1.7-2.8-fold on days 2, 7 and 14, respectively, the absence of changes in activity in the skin of period 1 on day 7.

Average increases for infusion of 1.0 mg / kg agalsidase beta:

1.8-, 4.8- and 3.5-fold for 1.0 mg / kg agalsidase beta (N = 2) on days 2, 7 and 14, respectively, increase in individual patients: 1,1- and 2, 5-, 3.6- and 6.0- and 1.7- and 5.4-fold on days 2, 7 and 14, respectively, an increase in the activity of α-Gal A in PBMC after co-administration of 0.5 or 1.0 mg / kg agalsidase beta and 150 mg migalastat are shown in FIG. 7-9.

Conclusion.

The interaction of 150 mg migalastat with 0.5 and 1.0 mg / kg of agalsidase beta led to an increase in the activity of α-Gal A regarding:

AUC of α-Gal A in plasma in all patients (N = 6), α-Gal A in the skin in all patients on day 2 (N = 5), but only in 3 out of 5 patients on day 7, α-Gal A in PBMC in all patients on days 2, 7, and 14 (N = 6).

The interaction of 150 mg migalastat with 0.5 or 1.0 mg / kg agalsidase beta led to 2-4-fold increase in AUC activity of α-galactosidase A, 1.1-3.9-fold increase in the activity of α-galactosidase A in the skin on day 2 and 1.1-6.0-fold increases in the activity of α-galactosidase A in PBMC on days 2, 7 and 14 compared with agalsidase beta separately. On day 7 after co-administration, four patients had increased α-galactosidase A activity in the skin.

A dose of migalastat 150 mg increased the enzymatic activity of a half dose of agalsidase beta (0.5 mg / kg) better than a full dose (1.0 mg / kg) up to 24 hours after a dose in plasma, skin and PBMC; however, the opposite was true (1.0 mg / kg> 0.5 mg / kg) at 7 and 14 days after dosing in the skin and PBMC. A summary table of results is shown in FIG. 10.

Regarding agalsidase beta separately, all patients at all time points had increased α-Gal A activity in PBMC relative to the initial level, however, 2 patients had reduced α-Gal A activity in the skin on day 2 relative to the initial level after 0.5 mg / kg infusion.

Example 3. The dosage regimen for the treatment of Fabry disease using migalastat hydrochloride and agalsidase.

The following example is an update to the study described in Example 2. This example includes an additional subject for a total of seven subjects.

The purpose of this example is to evaluate the safety and PK of two doses of migalastat HCl (150 and 450 mg), co-administered with ERT (agalsidase), in men with a diagnosis of Fabry disease.

Ways.

This is an ongoing, open, nonrandomized, 2-stage, fixed-sequence study.

Stage 1 includes 3 periods.

Period 1: intravenous infusion of ERT separately.

Period 2: oral administration of migalastat HCL (150 mg) 2 hours before intravenous infusion of ERT (at the same dose as in period 1).

Period 3: oral administration of only 150 mg of migalastat HCl. Patients eligible to participate in the study: males aged 18 to 65 years with Fabry disease.

Inclusion criteria.

Body Mass Index (BMI) is 18-35.

Begin treatment with agalsidase at least 1 month before the dose.

Estimated creatinine clearance (CLcr)> 50 ml / min at screening.

Exclusion criteria.

Documented transient cerebrovascular accident, ischemic stroke, unstable myocardial infarction within 3 months prior to screening.

Clinically significant unstable heart disease.

Sensitivity to or concomitant therapy with imino sugars (e.g., miglustat, miglitol).

Subjects received their current dose and the regimen of taking only agalsidase beta with one infusion (0.5 or 1.0 mg / kg for about 2 hours), followed by oral administration of 150 mg of migalastat HCl 2 hours before agalsidase beta following infusion.

- 16 031874

Five of these seven subjects received 0.5 mg / kg every two weeks, and two of the seven subjects received a dose of 1.0 mg / kg every four weeks.

In step 2, a 450 mg dose of migalastat HCl will be studied.

Steps 1 and 2 will be repeated with individual subjects with ERT infusion of agalsidase alpha (0.2 mg / kg for about 40 minutes).

Samples.

Serial blood samples were taken up to 24 hours after dosing to determine the activity of α-Gal A in plasma and protein levels in each period. Blood samples for α-Gal A activity in peripheral blood mononuclear cells (PBMCs) were taken prior to dosing and on days 1, 2, 7 and 14 of each period. A puncture biopsy on the activity of α-Gal A in the skin was taken before dosing in period 1 and on days 2 and 7 during periods 1 and 2. PK parameters of plasma α-Gal A activity include Stax, T _max , AUC _ot , AUC _{o -inf} and t _^1/2. Pharmacokinetic parameters were calculated using standard non-compartmental procedures (WINNONLIN version 5.0 or higher).

The activity of α-Gal A in plasma, skin, and PBMC lysates was measured using fluorescence enzyme analysis using 4-methylumbelliferyl-α-D-galactopyranoside (4-MUG). The effect of α-Gal A on 4-MUG was measured in vitro after serial dilutions for dissociation of migalastat.

Western blot analysis of α-Gal A protein was performed with plasma samples using antibodies to human α-Gal A. To calculate the corresponding concentration of α-Gal protein in each sample, a calibration curve of rhα-Gal A (agalsidase) was prepared.

Safety parameters included side effects (AE), vital signs, clinical laboratory tests (hematological analysis, serum biochemical analysis and general urine analysis), electrocardiograms (ECG), medical examinations and the use of additional drugs.

Results: preliminary results are available for stage 1, periods 1 and 2.

Distribution and demographic data of patients: the activity of α-Gal A in plasma, skin and PBMC was evaluated for seven patients from stage 1, periods 1 and 2.

All 7 patients received separately agalsidase beta for a period of 1; all patients were co-administered 150 mg of migalastat HCl 2 hours before the start of administration of agalsidase beta during period 2.

Two patients (identified as subjects A and B) received an intravenous infusion of 1.0 mg / kg agalsidase beta lasting 2 hours.

Five patients (identified as subjects C, D, E, F, and G) received an intravenous infusion of 0.5 mg / kg agalsidase beta lasting 2 hours, with the exception of 1: subject E was infused 2 hours and 40 minutes for a period of 2, but during period 1 was infused for 2 hours

All subjects were males aged 44-61 with Fabry disease, body mass index (BMl) was in the range of 20.9-29.1 kg / m ^2, and the estimated CLcr was in the range of 54-88 ml / min. The genotype of each subject is shown in FIG. eleven.

Security.

To date, 12 adverse events (AE) have been reported, one of which is serious. Serious AE was a transient cerebrovascular accident (TIA) that occurred after a screening visit, but before the dose was moderate, required hospitalization and was considered by the researcher as unrelated to the study drug. TIA was eliminated without complications. All other AEs were of mild severity, while all were considered as unrelated to the study drug and most were eliminated without treatment. Three AEs in three different subjects are constant: atrial extrasystole, atrial flutter and edema of the lower extremities, all not related to the study drug. The activity of α-Gal A in plasma AUC (area under the curve) of the activity of α-Gal A in plasma, depending on the activity-time profiles, with respect to treatment with 1.0 mg / kg agalsidase beta alone and treatment with 1.0 mg / kg of agalsidase beta in combination with 150 mg of migalastat HCl (with average values and standard deviations included) is shown in FIG. 12 for subjects A and B. The combined treatment of 1.0 mg / kg agalsidase beta and 150 mg migalastat HCl resulted in 2.2- and 1.6-fold increases in α-Gal A activity in subjects A and B, respectively, compared with treatment using monotherapy 1.0 mg / kg agalsidase beta.

Plasma AUC (area under the curve) of plasma α-Gal A activity depending on activity profiles time for treatment with 0.5 mg / kg agalsidase beta alone and treatment with 0.5 mg / kg agalsidase beta in combination with 150 mg migalastat HCl (with mean values and standard deviations included) is shown in FIG. 13 for subjects C-G. The combined treatment of 0.5 mg / kg agalsidase beta and 150 mg migalastat HCl resulted in 2.0-4.2-fold increases in αGal A activity compared to treatment with monotherapy with 0.5 mg / kg agalsidase beta.

Plasma α-Gal A activity when co-administered at all time points in most subjects increased separately with respect to enzyme replacement therapy (ERT), with the exception of one. Subject E received an unbalanced infusion, 40 min longer during period 2,

- 17 031874 which caused a relative decrease in enzyme activity during the infusion phase. However, in subject E, the activity at all time points after the peak of activity was increased with respect to ERT separately. Additionally, in accordance with Eng et al., Am.J. Hum. Genet. 68: 711-722 (2001) regarding α-Gal A activity, exposures increased nonlinearly.

Regarding co-administration with Migalastat HCl (period 2) compared to ERT alone (period 1), all subjects had increased plasma AUC activity of α-Gal A, as shown in FIG. 12 and 13. The average increase in plasma AUC activity of α-Gal A was 3.0-fold for 0.5 mg / kg agalsidase beta. Excluding a 2.0-fold increase in a patient with an unbalanced infusion duration, the average increase was 3.2 times. The average increase in plasma AUC activity of α-Gal A was 1.9-fold for 1.0 mg / kg agalsidase beta.

Α-Gal A activity in the skin.

In FIG. 14 shows that treatment of subjects with 0.5 or 1.0 mg / kg of agalsidase beta in combination with 150 mg of migalastat HCl increased the activity of α-Gal A in skin samples on day 2 compared with treatment with enzyme monotherapy alone. In FIG. Figure 15 shows the activity of α-Gal A in skin samples on day 7 after monotherapy and combination therapy with agalsidase beta and 150 mg migalastat HCl. The following average increases in α-Gal A activity in the skin were observed: 2.6-fold and 1.4-fold on days 2 and 7, respectively, for 0.5 mg / kg agalsidase beta (N = 5) and 1.9- and 1.5 -free on days 2 and 7, respectively, for 1.0 mg / kg agalsidase beta (N = 2).

The activity of α-Gal And in RMVS.

The following average increases in α-Gal A activity in PBMC were observed: 2.4-, 1.9-, and 2.1-fold on days 2, 7, and 14, respectively, for 0.5 mg / kg agalsidase beta (N = 5) and 1.8-, 4.8-, and 3.5-fold for 1.0 mg / kg (N = 2) agalsidase beta on days 2, 7, and 14, respectively.

Western blot analysis.

For 5 out of 7 subjects, changes in the plasma α-Gal A protein using Western blot analysis of the AUC period 2 (co-administration) / period 1 (ERT separately) were not observed. However, 2 subjects (subject C, which received 0.5 mg / kg agalsidase beta, and subject G, which received 1.0 mg / kg agalsidase beta) had a 20% increase in protein after co-administration compared to ERT alone (data not shown).

Conclusion.

The interaction of 150 mg of migalastat with 0.5 and 1.0 mg / kg of agalsidase beta led to an increase in the activity of α-Gal A in plasma, skin and PBMC. Co-administration of 150 mg of migalastat HCl and agalsidase beta was generally safe and well tolerated.

The scope of this application is not limited to the specific embodiments described herein. Various modifications to the application in addition to those described herein will, of course, become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

Additionally, it should be understood that all values are approximate and are for description.

This application contains patents, patent applications, publications, product descriptions and protocols, the disclosures of which are incorporated herein by reference in their entirety for any purpose.

Claims (13)

CLAIM 1. A method of treating Fabry disease in a subject, comprising administering from about 50 to about 600 mg of 1-deoxyhalactonojirimycin and an effective amount of a therapeutic agent to replace a deficiency of α-Gal A enzyme, characterized in that 1 deoxyhalactonojirimycin is administered over a period of from about 2 h up to about 30 minutes before the introduction of a therapeutic agent to replace the deficiency of α-Gal A enzyme, while the introduction of a therapeutic agent to replace the deficiency of α-Gal A enzyme follows the introduction of 1 deoxy laktonodzhirimitsina. 2. The method according to claim 1, characterized in that the amount of 1-deoxyhalactonojirimycin administered is from about 150 to about 450 mg. 3. The method according to claim 1, characterized in that the amount of 1-deoxyhalactonojirimycin administered is selected from about 150 mg, about 300 mg, and about 450 mg. 4. The method according to claim 1, characterized in that the patient abstains from eating for a period of time that begins about 0.5-4 hours before the administration of 1-deoxygalactone jirimycin and ends about 0.5-4 hours after administration 1 -deoxygalactone-jirimycin. 5. The method according to claim 4, characterized in that the patient refrains from eating for at least about 2 hours before the introduction of 1-deoxyhalactone-jirimycin and at least about 2 hours after the introduction of 1-deoxyhalactone-jirimycin. - 18 031874 6. The method according to claim 1, characterized in that 1-deoxyhalactonojirimycin is administered about 2 hours before the introduction of a therapeutic agent to replace the deficiency of α-Gal A enzyme. 7. The method according to claim 1, characterized in that 1-deoxyhalactonojirimycin is migalastat hydrochloride. 8. The method according to claim 1, characterized in that the therapeutic agent to replace the deficiency of αGal A enzyme is selected from agalsidase alpha and agalsidase beta. 9. The method according to claim 1, characterized in that 1-deoxyhalactonojirimycin is administered as an adjuvant for a therapeutic agent to replace the deficiency of the α-Gal A enzyme. 10. The method according to claim 1, characterized in that 1-deoxyhalactonojirimycin and a therapeutic agent to replace the deficiency of α-Gal A enzyme are administered as a combined therapeutic agent. 11. The method according to claim 1, characterized in that it is additionally administered a second dose of 1 deoxyhalactonojirimycin in the period from the moment the therapeutic agent is administered to replace the deficiency of the α-Gal A enzyme up to about 4 hours after that. 12. The method according to claim 6, characterized in that 1-deoxyhalactonojirimycin and a therapeutic agent to replace the deficiency of α-Gal A enzyme is administered every 1-4 weeks. 13. The method according to p. 12, characterized in that 1-deoxygalactone-jirimycin and a therapeutic agent to replace the deficiency of α-Gal A enzyme is administered every 2 weeks.